1. health and fitness
2. disease

Proteins/Enzymes
Wednesday, July 31, 9:30 am – 5:00 pm
treatment is required. In contrast, intracerebral haemorrhagic stroke (ICH) is
characterised by cerebral blood vessel haemorrhage leading to bleeding in to the
cerebrum resulting in damage. D-dimer is a fibrin degradation product and due to
the important role that fibrin plays in the clotting process during stroke, levels of
D-dimer may be important in identifying patients with stroke and predicting their
outcome. This study reports the correlations between plasma levels of D-dimer and
stroke severity and mortality in acute stroke patients, measured using Randox biochip
array technology.
Wednesday, July 31, 2013
Poster Session: 9:30 AM - 5:00 PM
Proteins/Enzymes
B-151
Identification of Novel Inflammatory Biomarkers in the Early
Diagnosis of Chronic Kidney Disease
M. Summers, M. Browne, C. Ledgerwood, C. Richardson, R. I. McConnell,
S. P. Fitzgerald. Randox Laboratories Limited, Crumlin, United Kingdom
Introduction: Chronic Kidney Disease (CKD) can go undiagnosed due to its
asymptomatic nature and is described as a progressive loss in renal function leading to
end-stage renal failure and death. In order to classify patients with early CKD (Stage
1), Modification of Diet in Renal Disease (MDRD) guidelines suggest an eGFR ≥ 90
ml/min/1.73m2 (based on the measurement of serum creatinine), with other evidence
of kidney disease (proteinuria, haematuria, kidney inflammation). The complexity in
diagnosing a patient with CKD at an early stage of disease has led to most patients not
receiving a diagnosis until the disease has progressed to an advanced stage.
Relevance: There currently are no accepted methods for easily determining kidney
disease at early stages. Inflammation plays a key role in the development and
progression of CKD and identification of inflammatory markers in patients suspected
of having CKD may play a useful role in the diagnosis of disease. Inflammation can be
monitored through the presence of signalling molecules, such as cytokines, and their
soluble receptors. This study aimed to identify soluble cytokine receptors; soluble
tumor necrosis factor 1 (sTNFRI) and 2 (sTNFRII) as potential novel markers to aid
diagnosis of CKD at early stages following a multi-analytical approach.
Methodology: Serum samples were taken from 327 patients with CKD (137 Stage
1, 109 Stage 2 and 81 Stage 3) and 139 healthy controls. Concentrations of sTNFRI
and sTNFRII in samples were determined using a cytokine biochip array applied to
the Evidence Investigator analyser. Serum creatinine was also measured to determine
eGFR using the MDRD method. Statistical analysis was performed using SPSS v20
(IBM), all data represented as Median [Range].
Results: Differences in concentration of each analyte across the disease groups were
initially assessed using the non-parametric Krukal-Wallis testing: both analytes were
shown to have significantly different levels across the different stages of disease
(significance determined as p<0.05). Post hoc analysis was performed comparing
CKD groups with controls, using Mann-Whitney (with Bonferroni correction): both
analytes demonstrated a significantly higher median concentration of the respective
analyte at all stages of CKD (Stage 1-3) compared to control. sTNFRI was shown to be
significantly increased at all CKD stages (0.6 [0.03-2.17], 0.73 [0.23-1.76],1.2 [0.513.88] ng/ml respectively; p<0.0001 for all) compared to control (0.44 [0.13-0.79]ng/
ml). sTNFRII was also shown to be significantly increased at all CKD stages (0.64 [03.21], 0.77 [0.17-3.23],1.44[0-10.09] ng/ml respectively; p<0.0001 for all) compared
to control (0.33 [0-0.92] ng/ml). Furthermore, the concentrations of both markers
were significantly increased at Stage 3 compared with Stages 1 and 2 (p<0.0001).
This was confirmed by correlation analysis between sTNFRI/II with disease stage (as
clinically determined) and eGFR. Both markers significantly correlated (p<0.0001)
with disease stage and eGFR.
Conclusions: This investigative study found elevated levels of sTNFRI and sTNFRII
in the serum of CKD (stages 1-3) patients compared to controls. Both markers
presented a significant correlation with disease stage and eGFR. These findings
indicate the potential utility of these inflammatory markers in diagnosing CKD at an
earlier stage as well as potential disease stratification markers.
B-152
Plasma Levels of D-Dimer as an Indicator of Severity and Mortality in
Acute Stroke: Application of a Biochip Array Kit
C. McKeever1, K. Makris2, C. Ledgerwood1, K. Kenwell1, C. Richardson1,
J. V. Lamont1, R. I. McConnell1, S. P. Fitzgerald1. 1Randox Laboratories
Limited, Crumlin, United Kingdom, 2Clinical Biochemistry Department,
KAT General Hospital, Athens, Greece
Background: Blood clots play a pivotal role in the pathogenesis of acute stroke.
Ischaemic stroke (IS) is characterised mainly by a thrombosis occluding the cerebral
arteries leading to ischaemia in the occluded region, consequently thrombolytic
Methodology: In a prospective study 98 patients with acute stroke were included
(73 IS and 25 ICH). The mean age (SD) of the patients was 75.2 (9.4) years. Stroke
severity was measured at the time of admission with the Scandanavian Stroke Scale
(SSS). Functional outcome was measured with the modified Rankin scale (mRS) on
day 7 and acute stroke patients were categorised into three severity groups (mild,
moderate and severe) according to their mRS-score: mild (mRS-score:0-2), moderate
(mRS-score:3-4) and severe (mRS-score:5-6). Blood samples were taken at the
time of admission and at 24, 48 and 72 hours thereafter. A final measurement was
performed on day 7. Forty-two patients (42%) died during a follow-up period of 1
year. The mean time (SD) between the onset of neurological symptoms and hospital
admission was 3.22 (1.58) hours. Sixty healthy subjects served as controls. D-dimer
levels were quantified in EDTA plasma samples employing a biochip array kit on the
Evidence Investigator analyser.
Results: At admission, mean D-dimer levels were significantly elevated in both IS
(343.4ng/ml) and ICH (540.8ng/ml) when compared to healthy controls (110.1ng/
ml) (p<0.0001 anova test). The diagnostic accuracy of a single D-dimer measurement
upon hospital admission for diagnosis of stroke was high [AUC=0.87 (95%CI 0.810.93), P<0.0001]. Mean D-dimer levels increased with severity and this biomarker
pattern was apparent upon admission (mild=256ng/ml, moderate=415ng/ml,
severe=555ng/ml) (p=0.011 anova-test). Plasma levels increased during follow-up
peaking at 7 days for both stroke subtypes (1059ng/ml for ICH and 363ng/ml for IS).
Mean D-dimer levels were significantly increased among non-survivors (317ng/ml)
compared to survivors (213ng/ml) (p<0.0001) at 24 hours. This difference was also
observed for 48 and 72 hour time-points.
Conclusions: The presented data suggest that the determination of plasma levels of
D-dimer upon admission can facilitate stroke diagnosis and serve as a predictor of
severity and mortality. These results indicate that there is an association between low
levels of D-dimer and better outcome among acute stroke patients.
B-153
Identification of novel biomarkers of hemorrhagic stroke by integrating
bioinformatic and mass spectrometry-based approaches
E. Martínez-Morillo1, P. García Hernández2, I. Begcevic1, H. Kosanam1,
B. Prieto García2, F. V. Álvarez Menéndez2, E. P. Diamandis3. 1Samuel
Lunenfeld Research Institute, Joseph and Wolf Lebovic Health Centre,
Mount Sinai Hospital, Toronto, ON, Canada, 2Department of Clinical
Biochemistry, Laboratory of Medicine, Hospital Universitario Central
de Asturias, Oviedo, Spain, 3Department of Pathology and Laboratory
Medicine, Mount Sinai Hospital, Toronto, ON, Canada
Background: Hemorrhagic stroke (HS) is a significant cause of morbidity and
mortality worldwide. Deterioration of patients is common in the first few hours after
symptoms onset so it requires rapid diagnosis and prompt medical attention. Moreover,
differentiating between hemorrhagic and ischemic stroke (IS) is critical to determine
treatment options. Computed tomography (CT) scan remains the cornerstone for
diagnosis of HS. However, it requires hospital admittance of patients. A blood-based
diagnostic test to identify patients that should be referred for a CT scan would be of
great value. In the present study, we hypothesized that proteins specifically expressed
in the brain at high levels may be released and detected in the cerebrospinal fluid
(CSF) of patients with HS. The aims were: (a) select “brain-specific” proteins using
a bioinformatic approach; (b) develop selected reaction monitoring (SRM) assays for
candidate proteins; (c) quantify these proteins in CSF samples from patients with HS,
IS and controls.
Methods: Towards the first aim, the Human Protein Atlas (www.proteinatlas.org) was
used to select proteins (n=390) with high expression in brain cell types and low or
absent expression in other body cell types (referred as “brain-specific”). Based on the
Peptide Atlas (www.peptideatlas.org) and to avoid high-abundance plasma proteins,
only “brain-specific” candidates with high number of observations in the “Brain
proteome” and low or zero observations in the “Plasma proteome” were selected
(n=76). For SRM assay development, protein extract from brain tissue samples was
used to identify “proteotypic” peptides for candidate proteins. Peptide identification
was confirmed in three ways: 1) prediction of retention times using SRRCalc 3.0
CLINICAL CHEMISTRY, Vol. 59, No. 10, Supplement, 2013
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Proteins/Enzymes
Wednesday, July 31, 9:30 am – 5:00 pm
(Skyline software), 2) co-elution of at least 6 transitions per peptide, 3) comparison
of the observed fragmentation pattern with the fragmentation pattern displayed in
publicly available databases (SRM atlas and GPM database). Based on SRM collider
(www.srmcollider.org), three transitions per peptide were selected to generate unique
ion signatures (127 peptides corresponding to 68 out of 76 candidate proteins). An
EASY-nLC 1000 pump coupled to a TSQ Vantage (Thermo Fisher) were utilized
for analysis. For protein quantification, twenty-one age-matched CSF samples were
selected, including patients with HS (n=7), IS (n=7) and healthy controls (n=7).
Samples were collected within 96 hours of symptoms onset and S100B protein was
measured using a fully-automated electrochemoluminometric immunoassay (Roche
Diagnostics).
providing a rationale to the fact that to date there is no natural mutation within this
region of Pro reported since any changes within this crucial amino acid region would
lead to dire clinical circumstances and are not compatible with survival.
Results: S100B was significantly elevated (p<0.05) in the HS group, especially in
those patients with intraventricular hemorrhage and poor outcome (Glasgow Outcome
Score ≤3). Thirty-three out of 68 “brain-specific” proteins were not detected in any
of the CSF samples analyzed. For remaining 35 proteins the coefficients of variation
from duplicates were <20%. Eight of these proteins (ENO2, GFAP, INA, MBP, MT3,
NEFM, SNCB, SNCG) were found to be highly elevated in samples from HS patients
(severe cases), with no elevations in the patients with IS and controls.
B. Jolly, S. M. Truscott. Beaumont Health System, Royal Oak, MI
Conclusions: Nine proteins, including some known biomarkers (ENO2, GFAP,
S100B), were found to be elevated in a subset of CSF samples from patients with
HS. Further verification and validation of these biomarkers of HS will be performed
in CSF and blood samples.
B-154
Amino acid sequence 473-487 of prothrombin is essential to inherent
coagulant activity
J. Wiencek1, J. Hirbawi2, M. Kalafatis1. 1Cleveland State University,
Cleveland, OH, 2Cleveland Clinic-Lerner Research Institute, Cleveland,
OH
The prothrombinase complex is the significant enzymatic complex that activates
prothrombin (Pro) to thrombin with rates compatible with survival. Prothrombinase is
composed of the enzyme factor Xa (fXa), and the cofactor factor Va (fVa) associated
on a procoagulant membrane surface in the presence of calcium ions. Suitable
prothrombinase formation results in the timely generation of a fibrin clot and the
arrest of bleeding due to the interaction of fVa with the members of prothrombinase
and a subsequent five-fold increase in the catalytic efficiency of fXa as compared
to the activity of fXa alone. Prothrombinase produces thrombin following two
sequential cleavages of Pro (at Arg320 followed by cleavage at Arg271) with formation
of an enzymatically active intermediate called meizothrombin. Although membranebound fXa is capable of activating Pro, the overall rate of thrombin formation is
not compatible with survival, and catalysis proceeds through the opposite pathway
(cleavage at Arg271 followed by cleavage at Arg320) with formation of prethombin-2.
fXa is known to exhibit a strong interaction with Pro in the presence and absence
of fVa. Previous studies have suggested that fXa interacts with Pro within amino
acid region 473-487 in a fVa-dependent manner. Thus we investigated the functional
importance of amino acid region 473-487 of Pro. We used site-directed mutagenesis
to construct a recombinant Pro molecule with the region 473-487 deleted (rProΔ473-487).
The deletion and wild type Pro (rProWT) were stably transfected in BHK-21 cells. The
two recombinant molecules were purified according to a well-established protocol
and during the last step Fast Performance Liquid Chromatography (FPLC) was used
equipped with a strong anionic Mono-Q 5/50 column and a calcium gradient was used
to isolate properly carboxylated rProΔ473-487 and rProWT. Both recombinant molecules
were assessed using gel electrophoresis to examine their ability to become activated
into enzymatic thrombin by fully assembled prothrombinase or fXa alone. The
recombinant molecules were also investigated for clotting and chromogenic activity.
Gel electrophoresis revealed that consumption of rProΔ473-487 by prothrombinase and
subsequent thrombin formation was considerably impeded when compared with
thrombin formation following cleavage of rProWT by prothrombinase. In contrast,
membrane-bound fXa alone, in the absence of fVa, exhibited a marked increase in
the rate of cleavage at Arg271 and activation of rProΔ473-487 as compared to cleavage
at Arg271 and activation of rProWT. Both recombinant proteins displayed a similar
cleavage pattern, implying that no major structural alterations took place in rProΔ473-487
following the mutation. Additionally, clotting assays revealed rProΔ473-487 was devoid
of clotting activity and severely impaired in its amidolytic activity while rProWT had
clotting and chromogenic activities comparable to human plasma-derived Pro. Our
data demonstrate that amino acid sequence 473-487 of Pro is necessary for optimal
rates of activation by prothrombinase. Also this investigation suggests that amino acid
region 473-487 is required for innate thrombin activity in coagulation. These data are
A216
B-155
Electrophoretic Identification and Isotyping of an Immunoglobulinbound Alkaline Phosphatase Macroenzyme Complex in a Patient
Serum
Background. Macroenzymes, or enzymes in the circulation bound in a highmolecular weight complex, can cause long-term elevation in the measured serum
activity of clinically relevant enzymes, such as creatine kinase or alkaline phosphatase
(AP). Most often, macroenzymes consist of enzyme bound to immunoglobulins (type
I), but may also form by binding to other proteins or cellular membrane fragments
(type II). While weak disease associations (e.g., autoimmune) have been described,
it is generally believed that detection of a macroenzyme should not be of clinical
concern. Macroenzymes are uncommon but important to recognize so that expensive
or harmful diagnostic testing can be avoided. The objective of our study was to
characterize in a patient serum a potential type I AP macroenzyme using the same
electrophoresis-based platform currently in service for AP isoenzyme quantitation.
Case. A 73-year old male was evaluated by his primary care physician, who ordered
a liver function test panel. AP was elevated (137 U/L; RI: 30-110 U/L), but all other
liver enzyme concentrations were within reference intervals. AP isoenzyme analysis
revealed an atypical isoenzyme pattern – weak staining of bone and liver isoenzymes
with unusually intense and diffuse staining in the region of the gel where intestinal
AP bands normally migrate. In order to distinguish an innocuous AP macroenzyme
from a possible true elevation of intestinal AP, which would suggest a more serious
condition, we considered possible approaches with the available technology.
Methods. AP isoenzyme quantitation was performed using Sebia® ISO-PAL agarose
gels and reagents with the Sebia Hydrasys electrophoresis system. The method
quantifies bone, liver, and intestinal AP isoenzymes after electrophoresis under
alkaline conditions. Bands are visualized using a chromogenic substrate. Patient
sera are applied to the gel in duplicate. In the first lane, bone and one of the liver
isoenzymes co-migrate, but are separated in the second lane by direct gel application
of a lectin that binds preferentially to the highly sialated bone isoenzyme and prevents
its migration. We hypothesized that an immunoglobulin-bound macroenzyme might
be detected if we replaced the lectin with antiserum to human immunoglobulin.
The index patient and a control patient were electrophoresed again by the ISO-PAL
method, but with and without antisera to human IgG, IgA, IgM, Ig-kappa, or Iglambda (Sebia Immunofixation reagents).
Results. The antisera did not change the migration of any AP isoenzymes in the
control patient. In the index patient, antisera to IgA, IgM, and Ig-kappa did not change
the migration pattern of the putative macroenzyme. The electrophoretic pattern was
unchanged compared to untreated lanes in the same gel. However, application of
either IgG or Ig-lambda antisera inhibited the migration of the questionable band,
which also became narrower and less diffuse, indicating that the antisera bound to
the macroenzyme.
Conclusions. Replacing the lectin with antisera to human immunoglobulin in
the ISO-PAL method effectively confirmed the presence of a type I macroenzyme
and also allowed for isotype determination. This approach is more specific than
protein precipitation with polyethylene glycol and does not require size-exclusion
chromatograpy to characterize high-molecular weight complexes.
B-156
Evaluation of Dried Blood Spots for Use in Isoelectric Focusing
Electrophoresis for Alpha-1-Antitrypsin Phenotype Interpretation
M. M. Duran. Geonostics, Lincolnshire, IL
Background: Laboratory diagnostics contribute significantly in the diagnosis
of Alpha-1-Antitrypsin (AAT) deficiency, utilizing AAT serum concentration,
AAT phenotype determination by isoelectric focusing (IEF) electrophoresis, and
genotyping. Dried blood spots (DBS) is a potentially attractive sample type for IEF
phenotype analysis on the Sebia Hydrasys because of the ease of sample collection.
Here we present a novel methodology for AAT phenotype determination from DBS.
Methods: Eighteen whole blood samples from known phenotypes of MM, MS, and
MZ were spotted on filter paper using 50 uL of sample. The blood spots dried overnight
CLINICAL CHEMISTRY, Vol. 59, No. 10, Supplement, 2013
Proteins/Enzymes
Wednesday, July 31, 9:30 am – 5:00 pm
and then the 50 uL dried blood samples were punched and rehydrated with a buffer/
deionized water solution. Ten uL of each extracted DBS sample was applied to the
IEF comb. Eighteen serum samples of the corresponding DBS phenotypes were also
placed on a comb for comparison. IEF was performed on the Sebia Hydrasys using
standard protocol and the resultant gel was stained, washed, and digitally scanned.
Results: In Figure 1, phenotype MM is on the left, phenotype MS is in the center,
and phenotype MZ is on the right. For each phenotype, the DBS sample is on the left
and the corresponding serum sample is on the right. All 18 DBS phenotype samples
displayed the unique identifiable banding patterns present in the serum samples.
P = 0.01. The mean serum h-sCRP on the third day for patients with fractures(but
without surgery) was higher (381.6 ± 122.7mg/L) than for those with soft tissue
injuries 376.1 ± 143.3mg/ L, and for controls 69.4 ± 68.1mg/L, P = 0.01.
Conclusion: C - reactive protein levels increase in the blood of trauma patients as a
result of tissue damage but decreases after the third day following trauma. However,
in the presence of infection, the increase was sustained. We therefore recommend that
serial quantitative c - reactive protein measurements be done as an adjunct to surgical
care in patients with acute injuries.
Conclusion: IEF of DBS samples adequately reveal the M, S, and Z alleles on the
Sebia Hydrasys. Work is underway to validate the 100+ additional rare alleles and to
establish AAT protein stability on the filter paper.
B-158
A Novel Immunoassay for the Determination of the Chemokine
RANTES
M. F. Kelly, V. Toner, I. Vintila, C. Ledgerwood, R. I. McConnell, S. P.
Fitzgerald. Randox Laboratories Limited, Crumlin, United Kingdom
Introduction: RANTES (CCL5/regulated on activation, normal T cell expressed and
secreted) is a member of the CC subfamily of chemokines. RANTES mediates the
trafficking and homing of classical lymphoid cells such as T cells and monocytes,
but also acts on a range of other cells, including basophils, eosinophils, natural killer
cells, dendritic cells and mast cells. A wide range of inflammatory disorders and
pathologies have been associated with increased RANTES expression, including:
asthma, allogeneic transplant rejection, atherosclerosis, arthritis, atopic dermatitis,
delayed-type hypersensitivity reactions, glomerulonephritis, endometriosis, some
neurological disorders such as Alzheimer’s disease and certain malignancies. It also
plays a key role in the immune response to viral infection. Current available immunoanalytical methods for the determination of this chemokine require sample dilution
prior to assessment, which could lead to inaccuracy in the pre-analytical steps as high
dilutions and small sample volumes are involved. This would be detrimental for the
final outcome of the analysis.
B-157
High Sensitive C-reactive Protein in Patients With Acute injuries
S. E. Idogun, E. Ayinbuomwan, P. E. Irhibhogbe, S. Ayinbuomwan.
University of Benin Teaching Hospital, BENIN, Nigeria
Background: For many years, C - reactive protein is known as a highly sensitive but
non-specific marker for acute inflammation.
Aim and Objectives: The aim of the study was to determine the serial serum level of
high sensitive C-reactive protein (h-sCRP) in patients with trauma.
Subjects and Methods: Subjects were patients who were involved in vehicle crashes,
burns, falls or other traumatic cases. A structured questionnaire was administered to
all subjects to document their ages, sex, occupation, date and time of trauma, date of
admission, duration of hospital stay and complications. Samples were collected from
participants for estimation of h-sCRP on days 1, 3, and 8. Samples were taken from
control subjects for one time estimation of CRP by photometric method.
Results: A total of 120 patients were studied, males were 98 (81.7%) and 22(18.3%)
females. The most frequent cause of trauma amongst the patients was vehicular
crashes 82(68.3%) followed by gunshot injuries 21(17.5%). The mean serum h-sCRP
of the patients on the first day was (165.8 ± 104.2mg/L) but it peaked on the third day
post trauma (378.8 ± 133.0mg/L) and declined on the eight day to 300.0 ± 156.5mg/L,
Relevance: This study reports the development of a biochip based immunoassay
for the determination of RANTES in neat samples, thereby eliminating the need of
sample dilution, which not only simplifies the analytical process but also contributes
to a more accurate determination.
Methodology: A chemiluminescent biochip based immunoassay was employed: a
TAG -specific monoclonal antibody was immobilised and stabilised on the biochip
surface. Reagent containing the RANTES specific antibody coupled to the TAG
and calibrator/sample were added to the biochip, which is also the vessel of the
immunoreaction. After incubation of 1 hour at 37°C and a washing step, a detector
antibody was added. After incubation of 1 hour at 37°C and a washing step, the signal
substrate was added. The chemiluminescent signal was detected by digital imaging
technology in the Evidence Investigator analyser. The system incorporates dedicated
software for data processing and archiving. The signal is directly proportional to
the concentration of the analyte in the calibrator/sample. Six matched serum, EDTA
plasma and platelet poor plasma samples from apparent healthy subjects were
analysed.
Results: Initial evaluation showed that the immunoassay generated a calibration
curve for RANTES spanning the range of 0-2000ng/ml. The functional sensitivity of
the assay was determined to be 9.2ng/ml with intra-assay (n=20) precision < 6%. The
average RANTES concentration in serum was 332ng/ml while the concentrations in
EDTA and platelet poor plasma samples were 104 and 124ng/ml respectively.
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Conclusions: These results demonstrate a new immunoassay method for the
quantification of RANTES in serum, EDTA plasma and platelet poor plasma without
sample dilution. This will improve the performance of the assay and increase the ease
of use by the end user. In addition, the assay is suitable for multiplexing technology
for which most immunoassays do not require sample dilution.
B-159
Development of Highly Specific Monoclonal Antibodies to FABP1
A. Gribben, P. Ratcliffe, P. Stewart, S. Savage, P. Lowry, R. I. McConnell,
S. P. Fitzgerald. Randox Laboratories Limited, Crumlin, United Kingdom
Introduction: Fatty acid binding proteins (FABP) are small cytoplasmic lipid binding
proteins that are expressed in a tissue specific manner. FABPs bind free fatty acids,
cholesterol, and retinoids; and are involved in intracellular lipid transport. Circulating
FABP levels are used as indicators of tissue damage. Some FABP polymorphisms
have been associated with disorders of lipid metabolism and the development of
atherosclerosis. Liver-type FABP (L-FABP/FABP1) was one of the first FABPs to be
identified. FABP1 is a ~14kDa member of the fatty acid binding family of proteins
and it is mainly expressed in hepatocytes of the liver, but is also found in proximal
tubular cells of the kidney and in colonocytes and enterocytes (jejunal and ileal) of the
gastrointestinal tract. FABP1 is a sensitive biomarker for abdominal disease/injury,
kidney disease and liver disease due to its high tissue concentration and low plasma
concentration along with its relatively small size that results in its early release after
tissue damage.
better sensitivity and specificity for ischaemic stroke. Nucleoside diphosphate kinase
(NDKA) was originally identified because of its reduced mRNA transcript levels in
highly metastatic cells. NDKA was identified as having potential utility for stroke
detection.
Relevance: The purpose of this study was to develop simultaneous biochip based
immunoassays for the multi-analytical determination of GSTP1 and NDKA. The
analytical performance of the biochip immunoassays was evaluated. This is relevant
as a new analytical tool to facilitate an improved diagnosis and treatment.
Methodology: The simultaneous chemiluminescent sandwich immunoassays defined
discrete test sites on the biochip surface. The chemiluminscent signal was detected
by digital imaging technology on the Evidence Investigator analyser.The intensity of
the signal is proportional to the analyte concentration in the original sample. Serum
samples from stroke patients (n=20) and from age and sex matched controls (n=20)
were analysed. Equal variance t-test was used for statistical analysis.
Results: Evaluation of the analytical performance of the simultaneous immunoassays
showed that the immunoassays were target specific for both GSTP1 (assay range
0-400ng/ml) and NDKA (assay range 0-300 ng/ml).The assessment of serum samples
from stroke patients and age and sex matched controls showed significantly increased
values of both biomarkers in stroke patients when compared to controls (3.05 ng/ml
versus 0.88 ng/ml, p<0.007 for GSTP1 and 16.15 ng/ml versus 4.12 ng/ml, p<0.001
for NDKA).
Conclusion: The data indicates that the developed biochip based immunoassays are
applicable to the simultaneous determination of GSTP1 and NDKA. In this study a
significant increase in the levels of both proteins was observed in stroke patients when
compared to age and sex matched controls. This represents a useful analytical tool for
research and clinical applications related to the diagnosis of acute stroke.
Relevance: The aim of this work was to develop two highly specific monoclonal
antibodies to work as a sandwich pair for FABP1, which will be used as a tool in the
development of efficient immunoassays for application in clinical settings.
Methodology: The nine members of the FABP family were expressed as full length
recombinant proteins in E. coli. Sheep were immunized with the sequence for FABP1.
Lymphocytes were collected and fused with heteromyeloma cells. Supernatants from
the resulting hybridomas were screened for the presence of FABP1 specific antibodies
using the other eight members of the FABP family in negative selection ELISA based
assays. Positive hybridomas were cloned to produce stable monoclonal hybridomas.
The antibodies were purified and evaluated by direct binding ELISA to determine
their specificity for FABP1.
Epitope mapping of capture and detector/tracer antibodies was also completed using
overlapping peptides derived from FABP1. Biochip based immunoassays were
employed to evaluate the analytical performance of the antibody pair, the assay was
applied to the Evidence Investigator analyser.
Results: Analytical evaluation indicated that the monoclonal antibodies generated
were specific for FABP1, exhibiting <0.1% cross-reactivity for FABP2, FABP3,
FABP4, FABP5, FABP6, FABP7, FABP8, FABP9 and myoglobin. Epitope mapping
established that the capture and detector/tracer antibodies bind to N- and C-terminal
regions of FABP1 respectively. On a biochip platform, the FABP1 assay showed a
sensitivity value of 0.66ng/ml with an assay range of 0 - 400ng/ml.
Conclusion: The data shows that the developed monoclonal antibodies are highly
specific for FABP1 and do not cross-react with other members of the FABP family.
This creates a new analytical tool for the determination of FABP1 in clinical settings.
B-160
Development of Biochip Based Immunoassays as Multi-Analytical
Tools for Application to the Diagnosis of Ischaemic Stroke
J. Preece, C. Ledgerwood, S. Brockbank, P. Lowry, R. I. McConnell, S. P.
Fitzgerald. Randox Laboratories Limited, Crumlin, United Kingdom
Introduction: Currently stroke is the third leading cause of death worldwide.
In cases of acute ischaemic stroke tissue plasminogen activator (tPA) therapy can
be administered for thrombolysis if diagnosed within 3 hours of symptom onset.
Inappropriate administration of tPA can cause serious adverse effects including
intracranial haemorrhage leading to death. The availability of rapid and highly
sensitive assays that can complement existing CT scanning procedures to provide a
definitive diagnosis of ischaemic stroke and rule out patients who have not suffered
an ischaemic stroke is relevant for the administration of the appropriate treatment.
Glutathione S-transferases (GSTs) are a family of enzymes that play an important role
in detoxification and are categorized into four main classes (alpha, mu, pi, and theta).
Studies comparing the performance of glutathione S- transferase pi (GSTP1) to that
of 28 biomarkers relevant to stroke detection indicated that this protein presented
A218
B-161
Protein S100B in serum and urine may predict mortality after severe
traumatic brain injury
A. Rodríguez-Rodríguez1, J. Egea-Guerrero2, A. León-Justel1, J.
Enamorado-Enamorado2, E. Gordillo-Escobar2, Z. Ruíz de Azúa-López2,
J. Revuelto-Rey2, Á. Vilches-Arenas3, F. Murillo-Cabezas2, J. Guerrero1.
1
Department of Clinical Biochemistry, Virgen del Rocio University
Hospital, IBIS/CSIC/University of Seville, Seville, Spain, 2NeuroCritical
Care Unit. Virgen del Rocío University Hospital, IBIS/CSIC/University
of Seville, Seville, Spain, 3Department Preventive Medicine and Public
Health. University of Seville, Seville, Spain
Background: Many researchers have highlighted the correlation between S100B
protein serum levels and the severity of intracranial lesions and patient outcome. But
there is not much information about the value of S100B protein in urine. The aim of
this study was to evaluate the role of S100B levels in serum and urine as an early
predictor of mortality after severe traumatic brain injury (TBI).
Methods: During 12 month, 55 severe head injury patients were included. Clinical
variables collected were: sex, age, score on the Glasgow after resuscitation, CT
results, extracranial lesions, score on the Injury Severity Score, and the final diagnosis
of death / survival a month post-trauma. Samples of blood and urine were collected at
the time of admission, 24, 48, 72 and 96 hours after the trauma.
Results: 18.2% of patients died a within a month of the trauma. S100B levels (both
serum and urine) were significantly higher in those who died than in survivors. ROC
analysis showed that S100B protein determination at 24 hours after a severe TBI
can predict mortality (AUC: 0.958 to serum AUC: 0.778 for urine). The following
set points were established: 0.461 mg / L for serum, 0.025 mg / L for urine, with a
sensitivity of 90% in both cases and a specificity of 88.4% in the case of serum, 62.8%
in the case of urine.
Conclusion: S100B serum levels, as well as S100B urine levels, act as a sensitive and
specific biomarker for the early detection of mortality after severe TBI.
CLINICAL CHEMISTRY, Vol. 59, No. 10, Supplement, 2013
Proteins/Enzymes
Wednesday, July 31, 9:30 am – 5:00 pm
B-162
Can CSF indices predict the presence of oligoclonal bands in isoelectric
focusing gels ?
I. A. Hashim1, S. Hirany2, P. Kutscher2, J. Balko1. 1University of Texas
Southwestern Medical Center, Dallas, TX, 2Parkland Memorial Hospital,
Dallas, TX
Introduction: Analysis of cerebrospinal fluid Immunoglobulin G (IgG) indices and
oligoclonal bands (OCBs) is essential in the investigation of multiple sclerosis and
other central nervous system disorders. We examined the utility of CSF indices in
predicting the presence of oligoclonal bands. CSF indices predictive of and associated
with positive OCBs in gels are not known. Knowing the indices values associated with
presence of OCBs may be helpful when examining isoelectric focusing gels.
Method: Paired CSF and serum IgG and albumin levels as well as associated
oligoclonal bands results were collected over a two-year period. CSF IgG index,
synthesis rate, as well as localized synthesis were calculated. Values obtained were
correlated with findings of OCBs by isoelectric focusing electrophoresis. IgG and
albumin levels were measured using BN-II-Nephelometer (Siemens, USA) and
OCBs by isoelectric focusing (Sebia, USA). Analysis was performed according to
manufacturers’ instructions.
Results: 376-matched sets of results were obtained. IgG index ranged from 0.26 to
3.48 (median 0.53), IgG local synthesis ranged from -14.67 to 38.02 (median -0.72),
IgG synthesis rate ranged from -24.61 to 223.96 (median -1.99), and albumin index
ranged from 1.5 to 54.3 (median 5.0). 79 samples exhibited blood brain barrier
impairment as indicated by an albumin index > 9 and were excluded from the
analysis. 26.3 % of patients were positive for the presence of OCBs by isoelectric
focusing. There was good correlation between IgG index and IgG local synthesis,
and IgG synthesis rate (r= 0.71 and 0.68), respectively. In patients with intact blood
brain barrier, an IgG index greater than 0.7 predicted the presence of OCBs at 91 %,
whereas, IgG index levels below 0.7 predicted absence of OCBs at 92.7%. In patients
with blood brain barrier impairment, IgG local synthesis had a better correlation with
the presence of OCBs.
Conclusion: This study suggests that CSF Indices can be used to predict the presence
of oligoclonal bands in isoelectric focusing gels. In patients with an intact blood brain
barrier, IgG index >0.7 is predictive of a positive OCB in gels. Those with impaired
blood brain barrier, IgG local synthesis is a better predictor than IgG index.
B-163
Optimization of gradient chromatofocusing HPLC and comparison
with reversed-phase HPLC in the chromatography of proteins
D. J. Anderson, H. Jogiraju, K. K. Pedada. Cleveland State University,
Cleveland, OH
Background and Objective: Gradient chromatofocusing (GCf) is a HPLC technique
that generates linear pH gradients on weak anion-exchange columns (in the present
study) with low-molecular weight buffer components. The advantages of GCf are its
flexibility in generating pH gradients compared to conventional chromatofocusing,
higher resolution and narrower peaks compared to ion-exchange HPLC, and
separation based on the protein’s pI. The objective of the present study is optimizing
and evaluating GCf in protein analysis by: 1) developing a new approach in generating
smooth linear pH gradients (problematic in GCf techniques); 2) studying the effect of
the number of buffer components on peak width and resolution; and 3) comparing
performance of GCf with reversed-phase HPLC.
Methods: The innovation in linear pH gradient generation (pH 6.50 to 3.50 in 40 min,
1 ml/min on Waters DEAE 8HR, 1000Å, 4.6 x 100mm, 8μ) was accomplished by
adding a “bridging buffer” into both the aqueous basic application buffer [consisting
of 10 mM each of bis-tris methane (6.46) and 3-methyl pyridine (5.68), and 5 mM
acetic acid (4.76), in 5% methanol adjusted to pH 6.50 with ammonium hydroxide]
and the aqueous acidic elution buffer [consisting of 5 mM 3-methyl pyridine (5.68),
10 mM acetic acid (4.76) and 10 mM lactic acid (3.81)] (buffer component pKa given
in the parentheses). The bridging buffers were acetic acid (a normal component of the
elution buffer) in the application buffer and 3-methyl pyridine (a normal component
of the application buffer) in the elution buffer Proteins were separated according to pI
with this four-component buffer system and the results compared with that obtained
by a seven-component buffer system, which had more narrowly spaced pKa buffer
components. Additional components in the seven-component buffer system were 10
mM 4-methyl pyridine (6.02) and 10 mM pyridine (5.25) in the application buffer and
10 mM 4-chlorophenyl acetic acid (4.19) in the elution buffer. Bridging buffers were
5 mM acetic acid and 5 mM pyridine. Reversed-phase HPLC used a C4, Nest Group,
250mm × 0.3mm, 5u, 300Ǻ column, employing a 50 min linear gradient from 2% to
81% acetonitrile in 50 min at a flow rate of 7 uL/min.
Results: Addition of the bridging buffer components smoothed the irregularity that
usually occurs in the middle portion of the pH gradient. Half-height peak widths in
the seven-component buffer system were less for the proteins studied than the fourcomponent buffer system: β-lactoglobulin A (0.94 vs. 1.24 min), β-lactoglobulin B
(0.5 vs. 1.11 min), bovine serum albumin (0.7 vs. 2.69 min), conalbumin (1.4 vs.
1.78 min) and ovalbumin (0.46 vs. 0.71 min). Resolution increased by an average
of 59% for the greater component buffer system. GCf performed better compared to
reversed-phase HPLC, with an average decrease of peak width and average increase
in resolution by factors of 2.7 and 6.9, respectively.
Conclusion: The fundamental studies presented here advance the technique of ionexchange HPLC in protein analysis. Potential clinical applications of GCf include
hemoglobin analysis and discovery of disease markers in 2D-HPLC proteomic
techniques.
B-164
A new method for immuno-turbidimetric measurement of Calprotectin
in feces, plasma and other body-fluids.
L. A. Hansson1, A. M. Havelka2, C. B. Johansson3. 1Department of
Molecular Medicine and Surgery, Stockholm, Sweden, 2Department of
Molecular Medicine and Surgery Karolinska Institute, Stockholm, Sweden,
3
Department of Molecular Medicine and Surgery Karolinska Insitute,
Stockholm, Sweden
Background: The interest in calprotectin has been increasing during last few years
due to its potential as a non-invasive, cheap and sensitive marker for inflammation,
particularly for intestinal inflammation. Fecal Calprotectin might in the future be used
as a screening tool to exclude unnecessary colon investigations.
Currently, calprotectin is measured with commercially available enzyme-linked
immunosorbent assays (ELISA) and EliA (Fluoroenzyme immunoassay), which are
marketed by several manufacturers. At present, these methods are time-consuming
and used only in clinical laboratories. Moreover, determination of calprotectin in feces
requires often manual and long pre-analytical processing of the fecal samples, which
may lead to very long turn-a-round time for the calprotectin results.
We have validated a new immune-turbidimetric assay for determination of calprotectin
in combination with a fully automatic system for pre-analytical processing of fecal
samples in order to improve efficiency and generate shorter turn-a-round time for the
Calprotectin results.
Methods: A new particle-enhanced immune-turbidimetric assay (Gentian, Moss,
Norway) for determination of Calprotectin was validated. Fecal Calprotectin was
assayed on a Cobas c111 system (Roche AG, Basel Schweiz) .
Pre-analytical processing of fecal samples was performed with a fully automated
robotic system (SoniC, S2G Scandinavia) and turn-around time for reporting of
results was well within a working-day.
Results: Linearity was proven throughout the measuring range from 1 to 50 mg/L for
plasma samples and 50 to 2500 mg/kg for fecal samples. Within-run CVs for fecal
Calprotectin ranged from 2,2 - 9,6 %, for concentration range 50 - 700 mg/kg. Good
agreement was achieved in the comparisons between the Gentian-Calprotectin assay
and the commercially available ELISAs (Calpro AS and BÜHLMANN Laboratories
AG: slope range 1,08 - 1,38, R2 = 0,89-0,92).
Conclusions: The immune-turbidimetric Calprotectin assay was shown to be precise
and accurate with proven linearity over the measuring range. Good comparability
was obtained with other commercially available ELISAs. The automatization of
both pre-analytical processing of fecal samples and measurement of Calprotectin
concentration resulted in improved efficiency and significantly shorter turn-aroundtime for reporting the Calprotectin results.
CLINICAL CHEMISTRY, Vol. 59, No. 10, Supplement, 2013
A219
Proteins/Enzymes
Wednesday, July 31, 9:30 am – 5:00 pm
Methods: We carried out a performance verification study and a method comparison
study against five other Ferritin reagents on a Hitachi 917 analyzer.
B-166
Use of correlation equations with the reference method to harmonize
Alkaline Phosphatase results of routine methods
X. Qin1, C. Qian1, S. Xie1, Z. Qi1, X. Cheng1, L. Qiu1, J. Miller2. 1Department
of Laboratory Medicine, Peking Union Medical College Hospital, Beijing,
China, 2Department of Pathology and Laboratory Medicine,University of
Louisville Hospital, louisville, KY
Background: Some large health care systems have multiple instruments and methods
for the same analyte. The results of these disparate methods often vary. Patients
may have analyses performed by more than one method and it is desirable for those
results to be comparable. Alkaline phosphatase (ALP) is an important enzyme for
evaluating hepatic, biliary tract, and bone diseases. However, ALP results vary among
the methods in our system. We hypothesized that we could adjust each routine method
based on its correlation with the reference method to make all methods equivalent and
comparable to the reference method.
Results: The new assay showed the correlation coefficient over 0.99 against the
current assay from Denka Seiken even on the unique clinical samples such as EBV
IgG positive samples, Rheumatoid factor high positive samples, etc. It showed
prozone tolerance that is at least equivalent to the other reagents or even better. The
new assay showed the best performance in terms of the sensitivity and showed the
lowest detection limit and smallest CVs from within-run imprecision with samples
around the lower threshold (5 - 15 μg/L).
Conclusion: The new Ferritin assay from Denka Seiken showed the best performance
compared to the other Ferritin assays already marketed. The new assay showed
excellent precision, sensitivity and prozone for diagnosis of various disorders where
Ferritin level is either abnormally low or high without nonspecific reaction. This new
assay can give laboratories a more economical and flexible approach for Ferritin
determination.
Methods: We set up the International Federation of Clinical Chemistry (IFCC)
reference method for ALP at 37oC (Clin Chem Lab Med. 2011;49:1439). The accuracy
of this method was confirmed by acceptable results on samples from the 2011
external quality assessment scheme for reference laboratories in laboratory medicine
(RELA-IFCC) and imprecision was less than 1%. ALP was measured in duplicate
on 10 patient specimens on each of 4 days (n=40) by the Roche Modular, Olympus
AU5400, and Siemens Dimension methods, and compared with the reference method.
ALP activities covered the analytical range (10-741 U/L). Correlation between
methods was evaluated using the least squares regression analysis, and Pearson
correlation coefficients were determined. Routine method results were adjusted using
the correlation equation. The predicted bias and 95% confidence interval (CI) were
calculated according to CLSI EP9-A2.
Results: All three routine methods correlated well to the reference method (r2>0.99).
The regression equations of the Roche, Olympus, and Dimension methods (y) vs.
the reference method (x) were, respectively, y=0.923x+2.996; y=1.084x-2.806; and,
y=1.084x+2.286. The extreme value of the 95% CI of the predicted bias at the 400
U/L Medical Decision Level of ALP is -35.1 U/L (-8.8%), +34.3 (+8.6%), and +43.1
U/L (+10.8%), for the Roche, Olympus, and Dimension methods, respectively. These
all exceed the desirable bias of 6.4% based on biological variation. The corresponding
adjustment equations (solving the equations for x) were, respectively, x=1.083y-3.246;
x=0.9225y+1.919; and, x=0.922y-2.109. These adjustment equations were used to
convert the routine method raw ALP activity (y) to the equivalent reference method
result (x). After making these adjustments, the regression equations of the Roche,
Olympus, and Dimension methods (y) vs. the reference method (x) were, respectively,
y=0.9994x-0.008; y=0.9998x-0.666; and, y=0.99996x+0.005. The extreme value of
the 95% CI of the predicted bias at the 400 U/L decision level, after adjustment, were
-3.6 U/L (-0.9%), -7.3 U/L (-1.8%), and -2.8 U/L (-0.7%), respectively. The bias of
all three methods was within the desirable bias of 6.4%. The adjusted routine method
ALP results match the reference method results extremely well.
Conclusion: By making these adjustments to the three routine ALP methods they
all agree much more closely to the reference method results and to each other. This
strategy for harmonizing the ALP results produced by our routine methods allows
individual patient’s results to be more consistent when different methods are used at
different times.
B-167
Development of a new homogeneous immunoassay for Ferritin with
ultra-sensitivity
M. Kano, R. Tachibana, Y. Sato, Y. Ito. Research and Development
Department, Denka Seiken Co., Ltd., Niigata, Japan
Background: Because of wide variety of clinical significances of Ferritin in both low
such as iron deficiency anemia and high serum levels (a lower threshold is 12 μg/L
or below and an upper threshold is 300 μg/L or greater), assays for serum Ferritin
level are required to have a wide assay range with high sensitivity. In addition, serum
Ferritin levels may increase very high in some disorders such as hemochromatosis,
hemosiderosis, etc. Thus it is also important for assays for serum Ferritin to have good
prozone (high dose hook effect) tolerance for accurate and reliable measurements. We
developed a new latex particle-enhanced turbidimetric immunoassay for serum and
plasma Ferritin with ultra-sensitivity and excellent prozone tolerance. We compared
our new assay to other Ferritin assays already marketed including the one currently
available from Denka Seiken.
A220
B-168
An Evaluation of Specific Protein Assays on Mindray’s BS-2000M1
Clinical Chemistry System*
Y. Li, J. Zhuang, C. Chen, X. Zhang, J. Liu. Department of Laboratory
Medicine, Guangdong Provincial Hospital of Traditional Chinese
Medicine, Guangzhou, China
Background: Specific proteins are useful markers in the clinical laboratory for the
diagnosis of immunologic disorders and monitoring changes in the normal polyclonal
mixture of serum immunoglobulins. Mindray’s BS-2000M1 (Shenzhen Mindray BioMedical Electronics CO., LTD., P.R. China) is a high throughput clinical chemistry
system of 2200 tests/hour with ISE, and has a broad test menu including specific
proteins (Calibrators are traceable to ERM-DA470k).
Objective: The purpose of this study was to evaluate the performance of specific
protein immunoturbidimetric assays on the new Mindray’s BS-2000M1 clinical
chemistry system in comparison to the Cobas® 8000 clinical chemistry system
(Roche Diagnostics, Germany) using patient specimens received into the laboratory
for routine testing.
Methods: Five of the currently available BS-2000M1 serum protein
immunoturbidimetric assays were evaluated as part of this study. Evaluation protocols
for precision were based on CLSI EP-5A2 methods. Linearity protocol was based on
CLSI guideline EP6-A. Method comparison was evaluated using samples spanning
the dynamic range based on CLSI guideline EP9-A2.
Results: Total precision targets were met for all specific proteins, as well as withinrun targets. Total %CV’s ranged from 1.34-3.15%. Linearity met the Mindray claimed
dynamic range in all cases. Linear regression results from the method comparison
studies between the BS-2000M1 and the Cobas 8000 are presented in the table.
Method Comparison Results Between BS-2000M1 and Cobas 8000
Assay
Range (g/L)
N
Slope
Intercept
C3
0.19-1.54
62 0.96
-0.07
C4
0.010-0.480
62 0.96
0.00
IG-A
0.18-6.26
64 1.03
-0.16
IG-G
5.82-22.89
65 1.06
0.30
IG-M
0.19-3.69
65 1.08
-0.02
CLINICAL CHEMISTRY, Vol. 59, No. 10, Supplement, 2013
R
0.998
0.997
0.997
0.992
0.998
Proteins/Enzymes
Wednesday, July 31, 9:30 am – 5:00 pm
Conclusions: With respect to precision, linearity, and accuracy of serum protein
assays tested, Mindray’s BS-2000M1 clinical chemistry system produces results that
were consistent with Cobas 8000 clinical chemistry system.
* not yet available for in vitro diagnostic use in the US.
B-169
Development of candidate reference material SRM 2924 C-reactive
Protein Solution
E. L. Kilpatrick. National Institute of Standards and Technology,
Gaithersburg, MD
The goal of the current research is to produce a pure compound certified reference
material for C-reactive protein (CRP) to be used in the standardization of clinical
laboratory assays. The Joint Committee for Traceability in Laboratory Medicine
(JCTLM) maintains a database of higher-order reference materials for laboratory
medicine and in vitro diagnostics. Currently, there is no JCTLM-approved material
for pure CRP. The National Institute of Standards and Technology (NIST) has
procured candidate material to generate SRM 2924, C-reactive Protein Solution. The
material was prepared as a solution of approximately 0.5 mg of recombinant CRP in
a volume of 1 mL aqueous buffer (concentration = 0.5 mg/mL) and will be analyzed
for protein concentration as well as structural conformation and related attributes.
Preliminary analysis of molar mass was performed using two methods: matrixassisted laser desorption ionization time of flight mass spectrometry (MALDI-MS)
and electrospray ionization mass spectrometry (LC/MS) with deconvolution. The
molar mass determined by each method was in close agreement (MALDI-MS (-313
ppm, n=10); LC/MS - (21 ppm, n=8) with the sequence calculated mass of 23029
Dalton including known post-translational modifications. Initial purity assessment
was performed using sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE, n=10) indicating that only a single band was evident upon Coomassie
blue staining. Further analysis will include amino acid analysis by isotope-dilution
mass spectrometry for concentration assignment and density measurements by the
Lang-Levy method. Issuance of SRM 2924 will enable the generation of secondary
reference materials using higher order methods leading to advances in standardization
of CRP clinical measurements.
B-170
Alpha-defensin in synovial fluid as a new biomarker for the diagnosis
of periprosthetic joint infection
C. Deirmengian1, K. Kardos1, P. Kilmartin1, A. Cameron1, D. Chung1, K.
Schiller1, K. Birkmeyer1, G. Kazarian1, J. Parvizi2. 1CD Diagnostics, Inc,
Wynnewood, PA, 2Rothman Institute of Orthopedics, Thomas Jefferson
University Hospital, Philadelphia, PA
Background: Prosthetic joint infection (PJI) occurs after primary joint replacements
at a rate of 1.0-2.5% and increases to 2.0-5.8% in revision surgeries. When treating
a painful joint replacement, the ability to distinguish between septic and aseptic
failure of the prosthesis is critical, as the treatment for PJI necessitates unique
surgical strategies that aim to eradicate the organism. Currently, surgeons utilize a
wide spectrum of tests in the attempt to diagnose PJI, including local measures of
synovial inflammation (synovial fluid white blood cell count and differential, synovial
tissue white blood cell count), systemic measures of inflammation (serum C-reactive
protein level, erythrocyte sedimentation rate), radiologic tests (radiographs, bone
scan), and bacterial isolation techniques (Gram stain, culture). Each of these methods
individually has limitations for either sensitivity or specificity. It has been reported
that 10-20% of all confirmed infections cannot be confirmed via culture methods.
The failure of these tools to reliably diagnose infection, and the resulting clinician
disparity in practice, recently led the Musculoskeletal Infection Society (MSIS) to
publish a consensus definition of PJI, utilizing a combination of clinical data and six
of the above tests.
Methods: We assessed the ability of alpha-defensin to distinguish prosthetic joint
infection from aseptic inflammation utilizing a series of well-characterized samples
that were clearly defined by the MSIS criteria utilizing an alpha-defensin ELISA
(Hycult) modified for use with synovial fluid. The study included 23 aseptic samples
and 22 septic samples that were provided by the Rothman Institute. Receiver
Operating Characteristics (ROC) analysis was conducted to select the optimal
cutoff for the assay and its performance was established verses the MSIS criteria.
Additionally, the performance of each of the individual methods utilized in the MSIS
criteria was evaluated utilizing the recommended criteria.
Results: A total of 45 well-defined samples were used to establish the optimal cutoff
(7.7μg/mL) for the alpha-defensins using a ROC analysis that yielded an area under the
curve (AUC) of 1.0. The sensitivity (and 95% confidence interval) for alpha-defensin,
ESR, CRP, WBC count, PMN% and culture were 100% (84.6-100%), 95.5% (77.299.9%), 95.5% (77.2-99.9%), 95.5% (77.2-99.9%), 95.5% (77.2-99.9%) and 90.9%
(70.8-98.9%) respectively. The specificity performances were 100% (85.2-100.0),
78.3% (56.3-92.5%), 87.0% (66.4-97.2%), 95.7% (78.1-99.9%), 95.7% (78.1-99.9%)
and 95.7% (78.1-99.9%) respectively. There was significant separation between the
positive and negative populations for alpha defensin where the concentration of the
lowest positive sample was 2.44 fold higher than the highest negative sample.
Conclusion: The measurement of alpha-defensin levels provides a highly sensitive
and specific method to aid the diagnosis of PJI with improved performance compared
to the traditional methods utilized for the analysis of synovial fluid.
B-171
Performance characteristics of The NGAL Test™ using the Roche
cobas c501 analyzer
S. L. La’ulu1, B. B. Suh-Lailam2, K. W. Davis3, A. E. Tebo2, J. A. Straseski2.
1
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City,
UT, 2Department of Pathology, University of Utah, Salt Lake City, UT,
3
ARUP Laboratories, Salt Lake City, UT
Background: Neutrophil gelatinase-associated lipocalin (NGAL) is a biomarker that
may aid in diagnosis of acute kidney injury and shows promise as a biomarker for
lupus nephritis. The objective of this study was to evaluate the analytical performance
of The NGAL Test™ (BioPorto Diagnostics) clinical chemistry application using the
Roche cobas c501.
Methods: Imprecision was tested using manufacturer’s quality control material, 3
serum pools, and 2 urine pools. Two runs of duplicate testing were conducted daily
for 5 days. Interference studies and limit of detection were performed for both serum
and urine matrices. Dilution linearity was assessed using manufacturer’s high and
blank calibrators. Method comparison testing was performed using the NGAL ELISA
kit as the comparator method. Samples used for testing were paired urine and serum
specimens from patients suspected of systemic lupus erythematosus.
Results: Imprecision studies had total CV’s ≤ 5.2%. Hemoglobin concentrations
up to 1335 mg/dL, bilirubin concentrations up to 7.25 mg/dL, and triglyceride
concentrations (serum only) up to 2350 mg/dL had no effect on results within the
precision of the assay for both serum and urine samples. The limit of detection was
10.7 ng/mL for serum and 4.6 ng/mL for urine. The assay was linear over a measured
range of 2.3 to 4934 ng/mL with maximum deviations from target recovery ≤ 8.4%
and slope of 1.02. Method comparison of the c501 to the ELISA by Deming regression
and Bland-Altman plots are summarized (Table 1).
Conclusions: The NGAL Test™, performed with the Roche c501, had favorable
precision and linearity. No interferences were observed for hemolysis, icterus, or
lipemia. A negative bias was observed between the 2 methodologies with the bias
being more prominent in urine samples. Overall, The NGAL Test™ performed well
on the Roche c501 and provides an alternative to the laborious ELISA method.
Sample Type
Serum
Urine
N
110
109
Equation
c501 = 0.89 x ELISA + 5.94
c501 = 0.75 x ELISA - 27.16
r
0.682
0.745
Bias
-33.6
-107.8
B-172
Development and evaluation of the performance characteristics of a
new microfluidic immunoassay for Haptoglobin on FRENDTM System
S. Lee, E. Choi, C. Lee, J. K. Chang, S. Han. NanoEnTek, Seoul, Korea,
Republic of
Background: The FRENDTM System is a portable, automated FRENDTM cartridge
reader which is based on quantitative immunoassay technology capable of quantifying
single or multiple analytes in 6 minutes by measuring laser-induced fluorescence in
a single-use disposable reagent cartridge. Haptoglobin (HP) is an acute phase protein
whose serum concentration rises significantly during acute inflammation due to
causes including surgery, myocardial infarction, infections and tumors.
Objective: The objective performance of this study was to evaluate a Haptoglobin
immunoassay on a micro-fluidic platform (FRENDTM cartridge-system).
Method: Human serum samples were diluted off-line with provided kit 10,000
fold prior to analysis. Serum HP concentration was determined by immuno-
CLINICAL CHEMISTRY, Vol. 59, No. 10, Supplement, 2013
A221
Proteins/Enzymes
Wednesday, July 31, 9:30 am – 5:00 pm
fluorescence assay of HP on FRENDTM system. The assay was standardized to the
IFCC International Reference Preparation CRM470 (RPPHS) and the result was
converted to mg/dL with consideration of dilution fold. We studied the precision and
linearity of the FRENDTM Haptoglobin assay (NanoEnTek, South Korea), compared
it with another test. Limit of detection establishment and interference testing were
also performed. Three samples were measured with 4 replicates and 5 runs for the
precision test. The linearity range experiment was performed using 7 equally spaced
concentrations including blank was prepared by Clinical and Laboratory Standards
Institute EP-6 dilution methods. Thirty eight samples were tested for comparing the
NanoEnTek’s assay with the Cobas Integra 800 Haptoglobin assay (Roche, Swiss).
Limit of detection was established by 48 measurements of both blank and low level
samples (CLSI EP-17 guideline) and three endogenous interferents were tested as per
CLSI guideline EP-7.
Results: The FRENDTM - HP assay demonstrated acceptable imprecision of %CV
(<10%) in low, intermediate, and high level samples. The linearity of the assay was
found to be acceptable in the range of 0~400 mg/dL (r=0.997). A method comparison
between Roche’s assay and NanoEnTek’s HP assay was made (Passing Bablok fit; y
= 1.38x - 29.20; x, Roche; y, NanoEnTek; n=38; y range; 2.67~502.66 mg/dL). No
significant deviation from linearity was found in the comparison study. The limit of
detection of the assay was 14.11 mg/dL. No significant interference (<=10%) was
observed from bilirubin, Intra-lipid and total protein up to concentrations of 20 mg/
dL, 3 g/dL, and 12 g/dL respectively.
Conclusion: The NanoEnTek’s FRENDTM Haptoglobin assay represents a rapid,
accurate and convenient mean of measuring HP quantity in human serum on
FRENDTM system.
*Assay currently under development and not for clinical use.
**All trademarks are property of their respective owners.
B-173
VERIFICATION OF A FULLY AUTOMATED LATEX ENHANCED
TIA FOR ADIPONECTIN
H. Mizue, Y. Minakawa, Y. Sato, Y. Ito. Research and Development
Department, Denka Seiken Co., Ltd., Niigata, Japan
Background: Adiponectin is solely secreted by adipocytes and acts as a hormone
with anti-inflammatory and insulin-sensitizing properties. Some studies indicate
that measurement of blood adiponectin levels can give useful information in risk
assessment of coronary heart disease and diabetes.
Herein, we report development of a new immunoassay utilizing latex particles to
measure blood adiponectin levels. Our new assay is a fully automated assay to be
applied to general chemistry analyzers.
Methods: We evaluated the performance of our new assay on Hitachi 917, Roche
Cobas c501, Roche Integra 400 and Beckman AU640 and compared it with
commercially available ELISA kits for measurement of total adiponectin.
Results: Lower detection limit (N=10, average + 2.6SD) of our assay was 0.5 ug/
mL on Hitachi 917 and Cobas c501 and 1.0 ug/mL on integra 400 and AU640.
By measuring of 10 serial dilutions prepared from a 40 ug/mL sample, Excellent
linearity was observed up to 40 ug/mL on all analyzers. The primary measuring range
up to 40 ug/mL is enough to cover clinically important levels. CVs in within run
precision study (N=10) were below 2% at all the adiponectin levels tested (Table 1).
In the method comparison study, our new assay (on Hitachi917) revealed excellent
correlation against 2 commercially available ELISA kits (ex. intercept=0.12, r=0.99,
with 96 serum samples against Millipore EIA). Correlations between Hitachi917
and other analyzers were also very good (Table 1). However, slopes were variable
depending on the ELISA kits. That suggests test results are not consistent among
different commercial assays.
Conclusion: Our new assay does not require off-line sample pretreatment or presample dilution, and it can give results much quicker than the existing ELISA kits (ex.
800 tests/hour on Hitachi 917 chemistry analyzer). Our new assay allows a simple and
quick approach to quantify blood adiponectin levels.
A222
B-174
Chemically stabilized PCR & RT-PCR reagents for all-ambient assays
S. de los Rios, V. Liberal, R. Partha, P. Singhal, R. Muller. Biomatrica, San
Diego, CA
Background: The need for cold or frozen storage of diagnostic testing reagents
makes the use of these tests difficult in regions where there is either no or unreliable
access to freezers. A reliable and cost effective alternative has been developed to
stabilize diagnostic test components dry at ambient temperatures. We have developed
and tested the stability and functionality of dried-down, stabilized PCR and RTPCR reagents which are described in this study. We combined the test reagents with
proprietary biostability compounds and applied a simple air drying procedure.
Following accelerated aging studies at elevated temperatures, we performed both
PCR and RT-PCR reactions to assess the ability of the dried down reagents to perform
the desired reactions.
Methods: We have evaluated several different assays using similar components,
testing varying combinations of dried down reagents with increasing complexity. The
reagents were combined with proprietary stabilizers and dried down in single shot
reactions. The dried reagents were incubated at 45°C for various periods of time and
tested in intervals of 3 days, 3 weeks, and 3 months. At each time point, the dried
reaction mixtures were rehydrated with the appropriate components to complete the
PCR or RT-PCR assay and the reactions were tested. Depending on the assay, either
gel electrophoresis (for end-point PCR) or Ct and melt curve analysis (for qPCR) were
performed to assay the ability of the dried reagents to perform their indicated function.
Each condition was run in triplicate and compared to a frozen positive control.
Results: For both PCR and RT-PCR assays, we have shown a minimum of 1 year
stability at ambient temperatures (based on accelerated aging at 45°C) for all
components of both end-point and qPCR assays when dried down and rehydrated with
template alone and template plus reaction buffer. qPCR results indicate that stabilized
reagents can amplify the desired template within 1.5 Ct values of the positive control.
Conclusion: These results demonstrates that the dry down of PCR and RT-PCR
reagents with biostability compounds leads to high retention of both stability and
activity at ambient temperatures (and elevated temperatures) while non-protected
reagents fail to perform their desired function. Therefore, similar procedures can be
applied to PCR-based diagnostic assays to eliminate cold chain requirements and
simplify testing in places with lack of access to reliable cold storage as well as for
applications that benefit from cost effective shelf life and shipping approaches.
CLINICAL CHEMISTRY, Vol. 59, No. 10, Supplement, 2013